Various conventional approaches have been used to visualize the surface of a biological sample, e.g., DNA spots of a micro-array such as a DNA chip, protein bands in a one dimensional (1-D) or two dimensional (2-D) gel, etc. For example, a DNA chip is generally a rigid flat surface, typically glass or silicon, that may have short chains of related nucleic acids spotted, e.g., DNA spots, in rows and columns, i.e., an array, thereon. Hybridization between a fluorescently-labeled DNA and specific locations on the chip can be detected and analyzed by computer-based instrumentation. The information derived from the results of hybridization to DNA chips is stimulating advances in drug development, gene discovery, gene therapy, gene expression, genetic counseling, and plant biotechnology.
Hybridization to DNA chips can be monitored by fluorescence optics, by radioisotope detection, and by mass spectrometry. The most widely-used method for detection of hybridization employs fluorescently-labeled DNA, and a computerized system featuring a confocal fluorescence microscope (or an epifluorescence microscope), a movable microscope stage, and DNA detection software. Technical characteristics of these microscope systems are described in U.S. Pat. Nos. 5,293,563; 5,459,325; and 5,552,928, which are all incorporated herein by reference. Further descriptions of imaging fluorescently immobilized biomolecules and analysis of the images are set forth in U.S. Pat. Nos. 5,874,219; 5,871,628; 5,834,758; 5,631,734; 5,578,832; 5,552,322; and 5,556,529, which are all incorporated herein by reference.
In brief, these conventional approaches to visualizing the surface of a DNA chip involve placing the chip on a stage of a microscope, moving the stage to put the sample into focus with a microscope objective, and triggering a digital camera or similar device to capture an image. The microscope objective is generally a device made of a group of lenses that have a sophisticated design that collects light from the sample, magnifies the image of the sample, and minimizes the unavoidable image and color distortion caused by the passage of the light through the objective. The light collected from the sample may pass through the microscope objective and through a set of mirrors and lenses until it is delivered to an eyepiece or the camera. The light path is the path that the light takes from the point where it leaves the surface of the sample until it reaches an imaging device such as an eyepiece or camera. The microscope generally is associated with a light source that directs light onto the sample.
These microscopes also generally have sets of optical filters that allow for viewing of fluorescent images. For example, the DNA that is hybridized to the surface of the DNA chip is typically labeled with fluorescent molecules that absorb light at one wavelength and then emit a different wavelength. The microscope may be equipped with sets of optical filters that block the wavelengths of light from the light source associated with the microscope but which allow the light emitted by the fluorescent molecules to pass therethrough such that the light may reach the eyepiece or camera. The light source is typically integral with the microscope and is an important part of the imaging system.
Further, generally, 1-D and 2-D electrophoresis processes are electrophoretic methods for resolving complex mixtures of proteins, e.g., 2-D electrophoresis is a multiple step process for resolving complex mixtures of proteins. For example, in 2-D electrophoresis, the proteins in a sample may first be dissociated into polypeptide subunits by dissolving in an appropriate buffer. The sample may then be applied to the top of a tube gel containing ampholines. Isoelectric focusing (IEF) is then carried out in the first dimension. After the IEF is carried out, the gel which contains the peptides is removed from the gel tube and placed over a slab gel and sealed by overlapping stacking gel; and thereafter, the second dimension of slab gel electrophoresis is carried out (e.g., proteins (negatively charged) run from the top of the slab (held at a negative charge) toward the bottom of the slab (held at a positive charge)). Then, staining, e.g., using a stain that binds to proteins so they can be identified, and an optional drying step, is carried out. Thus, the polypeptides are separated according to the independent parameters of isoelectric point in the first dimension and molecular weight in the second dimension (e.g., protein bands). For example, a large number, e.g., 1000, polypeptide spots (e.g., protein bands) may be resolved on a single two dimensional gel. Further, for example, such proteins in a 2-D gel may be labeled with fluorescent or chemi-luminescent markers.
Such protein bands in 1-D and 2-D electrophoresis may also be visualized and analyzed. Like DNA chips, such visualization of the protein bands in the 2-D gel is generally performed using the same conventional approaches as used to visualize the surface of a DNA chip, e.g., a microscope with the sample put into focus using a microscope objective, a digital camera, etc.
These conventional microscopes are sophisticated and expensive instruments that require training and maintenance. A single microscope objective typically has multiple lenses that are generally very expensive. A lens generally refers to a transparent solid material shaped to magnify, reduce, or redirect light rays, e.g., focus light. A light filter or mirror is distinct from a lens. Furthermore, use of a microscope requires a dedicated workspace that is approximately the size of a typical desk. Conventional microscopes have a light path that is several centimeters long that transmits collected light through air and other assorted optical devices within the light path. One of the challenges in microscopy is making the microscope as efficient as possible in capturing all of the light that leaves the sample surface so that an optimal image can be captured.
This costly instrumentation conventionally used to image biological samples, e.g., DNA chips, impedes the broad usage of such technologies. Therefore, an inexpensive, low-maintenance alternative spot detection method and apparatus for biological sample analysis, e.g., DNA chip analysis, that is easy to use and requires a minimum of space and maintenance is needed.
Integrated electronic circuit arrays for light-detection (e.g., a member of a group of detectors referred to as electronic light detectors) are readily available. They generally are based on CCD (charge-coupled device) or CMOS (complementary metal oxide semiconductor) technologies. Both CCD and CMOS image detectors are generally two-dimensional arrays of electronic light sensors, although linear imaging detectors (e.g., linear CCD detectors) that include a single line of detector pixels or light sensors, such as, for example, those used for scanning images, are also available. Each array includes a set of known, unique positions that may be referred to as having addresses. Each address in a CCD or CMOS detector is occupied by a sensor that covers an area (e.g., an area typically shaped as a box or a rectangle). This area occupied by a single sensor is generally referred to as a pixel or pixel area. As used herein, a light-detecting sensor located in a pixel area is referred to as a detector pixel. A detector pixel, as generally used herein, may be a CCD sensor, a CMOS sensor, or any other device or sensor that detects or measures light. The sizes of detector pixels vary widely and may have a diameter or length of 0.2 μm, which is also the theoretical limit of resolution of a light microscope. It is noted that some detectors have resolving sizes lower than 0.2 μm, such as for use in microscopes employing light of wavelengths of 180 nm and below.
CCD detectors, widely used in consumer and scientific applications such as digital recorders and digital cameras, are sensitive, and may be made with detector pixels that are smaller than those of CMOS devices. CMOS devices are now beginning to be incorporated in recorders and cameras because they are less expensive to produce. CMOS devices also are easier to interface with external control systems than CCD detectors. Some readily-available CMOS devices are integrated with the capabilities to provide acquiring, digitizing, and transmitting an image without additional circuitry, while CCD detectors generally require additional circuit elements to accomplish the same tasks.